1. Field of the Invention
The present invention relates to a novel pyrylium salt useful for the detection of a nucleic acid by the use of an optical means. It also relates to a stain containing a pyrylium salt for use in the detection of a nucleic acid by the use of an optical means, and a detection method of a nucleic acid by the use of the stain. Furthermore, it relates to a stain for a nucleic acid containing a pyrylium salt useful for the detection of a hybrid by a hybridization method using a probe for the detection of a variation at a nucleic acid level, and a detection method of a nucleic acid by a hybridization method using the stain for the nucleic acid.
The present invention also relates to a staining method and an observation method for microorganisms such as bacteria, yeast, mold, algae and protozoan as well as cells, a tissue and a chromosome of an animal and a plant. In addition, it relates to a detection method of a nucleic acid by an in situ hybridization using a probe containing a pyrylium salt as a label for the detection and identification of a specific sequence of the nucleic acid in biological samples.
2. Related Background Art
As modes by which a substance can interact with the double helix of DNA, there are known a case where the substance Gets into the base pair of a nucleic acid as an intercalater, a case where the substance is buried in grooves of the double helix, and a case where the substance approaches so as to be contiguous with the double helix.
The above-mentioned intercalater is usually a lamellar compound having a spread electron cloud, and it is arranged on an extended line of the stacked base pairs of the nucleic acid, i.e., on the axis of the double helix at the same distance as the distance between the base pairs of the nucleic acid in parallel with the base pairs of the nucleic acid. When intercalated in DNA, the intercalater characteristically leads to the fact that an optical absorption spectrum shifts to a longer wavelength side, absorption intensity decreases, or fluorescent intensity increases. For example, a dyestuff, in which the fluorescent intensity increases more in the case that the interaction with DNA takes place (when intercalated) than in the case that the interaction with DNA of a free state or the like does not occur, is used for the various detection operations of the nucleic acids such as agarose gel electrophoresis by the utilization of its characteristics.
Examples of a well-known intercalater include acridine orange, proflavine, ethidium bromide, donomycin and actinomycin. In particular, ethidium bromide and acridine orange are well known.
However, most of these dyestuffs have excitation wavelengths in an ultraviolet region, and hence an intensive ultraviolet lamp must be used, so that special care is required in order to protect persons from the lamp. For example, at the time of the detection of DNA, some equipment is required to protect the eyes and skin of an operator from the ultraviolet rays, and such equipment is on the market. Furthermore, in order to take the photographs of the electrophoresis patterns of DNA, a device such as a transilluminator is usually used, but the exposure adjustment of a camera in this case cannot be directly seen by the naked eye and so it is an intricate operation of trial and error. Moreover, in the case that the cutting of DNA from the agarose gel is directly seen by the naked eye, the operator cannot be completely protected from the irradiation with the ultraviolet rays even by the employment of the protective equipment. In addition, in the detection of DNA in the state of a liquid by the utilization of the dyestuff which can be excited by the ultraviolet rays, the irradiation of the intensive ultraviolet is always continuously given, so that harmful ozone and the like are also generated. In a certain case, DNA itself might be damaged by the ultraviolet irradiation. Much attention is being paid to an in situ hybridization in which hybridization is carried out in live cells to detect the live cells, but in the case that the dyestuff which can be excited by the ultraviolet rays is used as a label, the cells themselves are damaged by the ultraviolet radiation for the detection on occasion.
On the other hand, in the fields of medicine, biology and the like, biological specimens of microorganisms, human cells or animal cells are observed by a microscope or another means for the purpose of research, diagnosis and the like, but for such an observation, staining is usually widely carried out. Staining methods which can be utilized in such a purpose can usually be classified roughly into two categories. In the first category, there is a staining method in which a specific component which can be utilized for the staining of a portion to be observed and present in the biological specimen is stained with a dyestuff. As principles of the staining, there are a case where a target is stained with the dyestuff itself and a case where a coloring component is deposited on the present position of a specific component through various chemical reactions. They can often be used for pathological tests, and various staining methods have been conceived for various target components. These staining methods mainly intend to make visible a specific component, a specific morphology or a specific region, and they are often used for the observation of relatively macroscopic targets.
On the contrary, the staining method of the second category is called a fluorescent staining method. In the fundamental operation of the fluorescent staining method, a fluorescent dyestuff is first introduced into a present site of a specific component to be stained. As an introduction technique, there is a case where a target specific affinity of the dyestuff itself is utilized (the staining of an nucleic acid with ethidium bromide), and a case where a substance having a target specificity such as an antibody is bonded to the dyestuff, and its specific affinity is utilized to introduce the dyestuff into the target to be stained. These fluorescent staining methods are often used in a research field such as biology. In the fluorescent staining method in which the target to be observed is fluorescent, the detection sensitivity of a signal is high, and precision is also higher than the above-mentioned staining method by the coloring, and the microscopic targets can be handled. In general, the main purpose of the fluorescent staining method is often to detect a specific component.
In either method of the usual staining method and the fluorescent staining method, the specimen which is the target to be stained such as cells is usually fixed with an aldehyde or the like prior to the staining. This has the effect of improving the permeability of the dyestuff for use in the staining or another reagent into the specimen, and the effect of preventing the specimen from breaking during various operations necessary for the staining. Furthermore, in addition to the fixing operation and the staining operation, a washing operation is required to remove the excessive dyestuff, reagent and the like. This is essential to decrease background and to obtain stable results at the time of the staining.
On the other hand, in recent years, as carcinogenesis mechanism and the metastasis mechanism of cancers have been elucidated, it becomes known that the translocation of a chromosome and the deletion of the chromosome are closely concerned with these mechanisms. That is, cellular cancer genes are those which take part in the adjustment of reception, transmission or transcription of a growth signal in normal cells, and it has been elucidated that the genes are activated as the cancer genes by the mutation or the abnormal amplification of this gene, the translocation of this gene to the neighborhood of the gene being vigorously transcribed, or the bond to another gene. Furthermore, it is considered that the deletion of a cancer inhibitory gene from the chromosome is also concerned with the carcinogenesis mechanism. In addition, also in various kinds of hereditary diseases, the gene deletion and the translocation have been made apparent. As described above, if the diagnosis by the detection of the abnormal gene at a chromosome level is possible, the growth of tumor can be inhibited by transfecting the normal chromosome into the abnormal chromosome, and the possibility of the remedy of the cancer and the hereditary disease by compensating the detection of the gene can also be expanded.
As a method for knowing the abnormal chromosome, there is FISH (fluorescence in situ hybridization). This method comprises observing a formation ability of a hybrid of a probe corresponding to a gene to be inspected and a gene of the chromosome, and at present, it is considered to be the most accurate method. In this method, the gene to be inspected is limited by the probe to be used, and so when the target to be detected is definite, this method can be directly utilized. However, in the case that it is intended to screen the whole chromosome, this method is not always suitable. Thus, the whole chromosome may be first checked, and sites, where abnormalities such as translocation presumable from an abnormal conformation or deletion presumable from an abnormal length takes place, may be then screened to focus the target on the abnormal site. Afterward, FISH can be utilized, whereby the precise genetic diagnosis is considered to be possible in a wide range.
In order to carry out such a screening, it is necessary to beforehand distinguish or identify the chromosome. As the distinction or the identification method of the chromosome, a differential staining method of the chromosome is utilized. The distinction of the chromosome by this differential staining method utilizes the fact that when the chromosome is dyed in a band state by the use of a specific dye or fluorescent dyestuff after various pretreatments, the relation between its distribution and density is constant for each chromosome and characteristics in each chromosome. This technique is utilized for the distinction of the chromosome or the detection of an abnormality.
As the usual differential staining method of the chromosome, there are known, for example, Giemsa staining (G band), quinacrine staining (Q band) and R band staining. In the Giemsa staining method, the strain of the higher-order structure of the chromosome which is caused by a trypsin treatment is differentiated and observed as the loose state of the chromosome. Furthermore, according to the staining with quinacrine which is a fluorescent dyestuff, the chromosome is stained in a deep and light striped pattern along its vertical axis, and this can be observed by a fluorescent microscope. This fluorescent pattern is inherent in each chromosome, and so the distinction or the identification of the chromosome can be achieved by this. The portion stained with quinacrine is a position where A-T pairs are rich, and the quinacrine staining is different from the above-mentioned Giemsa staining in a stained state. In the R band staining, chromomycin A.sub.3 which is a fluorescent dyestuff is bonded to G-C pairs, and then treated with distamycin A which is a non-fluorescent dyestuff specific to the A-T pairs. In this case, a portion where G-C pairs are rich is only stained in the form of an R band, and the staining is characteristically carried out in a deep and light state opposite to the Q band staining and the G band staining. In the Q band staining and the R band staining of these differential staining methods, the detection sensitivity of a signal is higher as compared with the G band staining method by a coloring method, and they are also effective to handle a more precise microscopic target.
In this differential staining method of the chromosome, the more precise distinction or identification of the chromosome becomes possible by increasing the kinds of stains specific to sites of the chromosome, and so it is important for the more precise distinction or identification of the chromosome to develop the stain for permitting the differential staining specific to a different site, in addition to the already existent reagents (stains) for the differential staining.
On the other hand, many salts of pyrylium or thiopyrylium compounds in which the 2, 4 and 6-positions of a pyrylium ring or a thiopyrylium ring are substituted by substituted or unsubstituted phenyl groups have been suggested. Most of these salts are desirable as recording media, and for example, the specification of U.S. Pat. No. 4,341,894 describes that they are useful as sensitizers for electrical photoconductive compositions. In a biological field, it is described in Japanese Patent Application Laid-open No. 59-133460 that a 2,4,6-triphenylpyrylium or thiopyrylium compound, or a compound in which one of the phenyl groups substituted on the pyrylium ring or the thiopyrylium ring of this compound is replaced with a substituted styryl group is used as a stain for staining cells in a biological specimen. Furthermore, in Japanese Patent Application Laid-open No. 1-153683, it is described that a compound in which at least two of these phenyl-substituted groups have amino groups has a good efficacy for the remedy of a cancer.
When a dyestuff having an excitation wavelength in an ultraviolet region is used for the detection of a nucleic acid, the above-mentioned problems take place, and therefore as a means for avoiding these problems, it can be considered to use a dyestuff which can be excited by visible light. However, in most of the visible light excitation type dyestuffs which have been now utilized for the staining the nucleic acid, stokes shift is as slight as 20-30 nm, and so they have the drawback that an a signal to noise (S/N) ratio is bad at the time of the detection. As employed herein "S/N" means signal to noise ratio.
In an agarose gel electrophoresis or the like, when the degree of the increase of fluorescent intensity at the time of interaction with a nucleic acid is larger as compared with a case of no interaction with the double stand nucleic acid, the S/N ratio at the detection is good and it is possible to enhance detection sensitivity. However, the visible light excitation type dyestuffs which can sufficiently increase the fluorescent intensity are not known at present.
As described above, in order to solve the problems which the ultraviolet excitation type dyestuffs have, the visible light excitation type dyestuffs have been largely desired which has the large stokes shift and the much higher fluorescent intensity at the interaction with the nucleic acid as compared with the case of no interaction.
Moreover, in the conventional staining method of biological specimen such as cells, operations such as the fixation of the specimen by the use of formaldehyde or the like and the washing/removal of the excessive dyestuff are essential to obtain reproducible and stable results.
For example, the operation of the specimen fixation is important to improve the permeability of reagents of the dyestuff and the like or to maintain the morphology of the obtained specimen. In addition, the dyestuff for the staining, another reagent and the like are usually used in large excess of the specimen. This has effects of the curtailment of an operation (staining) time and the like but simultaneously causes the high background. Therefore, the washing operation between the respective operations is important to decrease the background at the time of observation or measurement and to thereby obtain a more correct judgement. For these reasons, the washing operation has been elaborately carried out. However, these operations are intricate and take much time, and hence in the case that a large amount of the specimens are treated, they interfere with a prompt treatment operation.
Therefore, the omission or simplification of operations such as the fixation and the washing of the specimens which are intricate and take much time are strongly desired.
Moreover, most of the fluorescent dyestuffs which have been heretofore used give rise to a vigorous fade phenomenon, and after the irradiation of the excitation light, the fluorescence rapidly disappears. Accordingly, contrary to the usual staining, it is difficult to observe a fluorescent image for a long period of time, and the preparation of the permanent specimen by the fluorescence staining is almost impossible. For these reasons, it is a desired to develop a preparation method of the fluorescence-stained specimens which can be stored for a long period of time.
In the field of the differential staining of the chromosome, a stain which can obtain a stained pattern different from that given by the conventional stain is highly desired, as described above. In addition, as in the case of the above-mentioned staining of the biological specimen, also in the differential staining of a chromosome, operations such as the fixation of the specimen by aldehyde and the washing of the excessive stain take time and equipment, and they interfere with the prompt achievement of a treatment such as mass screening of a large amount of the specimens.